Bottom Line:
The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved.This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia.Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins.

Affiliation: Program in Neuroscience, Uniformed Services University of the Health Sciences , Bethesda, MD , USA.

ABSTRACTProtein citrullination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS), and prion disease. Citrullination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citrullination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up--regulated protein citrullination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders, including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically induced calcium excitotoxicity was shown to activate the citrullination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type, and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.

Mentions:
The regional effects of CCI on protein citrullination are summarized in Figure 3 and Figure S2 in Supplementary Material. CCI produced a marked increase in protein citrullination throughout the injured cortex, extending from lateral to the lesion site to regions of the cortex not directly impacted by CCI (Figure 3B). Scoring of this region revealed that male naïve rats (mean score: 0.06) and male CCI rats (mean score: 1.43) were significantly different, p < 0.001, and female naïve rats (mean score: 0.04) and female CCI rats (mean score: 1.35) were also significantly different, p < 0.001 (n = 11 male rats, CCI; n = 8 male rats, naïve control; n = 10 female rats, CCI; n = 7 female rats, naïve control) [F(3,32) = 31.05, p = 0.000, with post hoc Tukey HSD analyses considering injury and gender variables independently]. Similarly, immunolabeling of the injured ipsilateral hippocampal formation (Figure 3E) and external capsule (Figure 3H) showed similar increases in protein citrullination in these regions. To a lesser extent, protein citrullination was also observed in fibers extending ventrally from the lesion site toward the midline corpus callosum (not shown). Other brain regions, including the amygdala and caudatoputamen, were completely negative for protein citrullination in these CCI animals. Furthermore, scoring of the cortex revealed that there was no gender difference observed in the regionalization or magnitude of protein citrullination following injury (CCI: p = 0.968; naïve: p = 0.999). In general, CCI appeared to have little effect on the status of protein citrullination in contralateral brain structures, with the exception of the dorsal hippocampus, where ~20% of injured animals (4 of 21) displayed an intense labeling of unusually large and rounded cells (Figure 3F). Finally, 6B3 immunolabeling was uniformly low across all regions studied in control male and female animals (Figures 3A,D,G).

Bottom Line:
The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved.This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia.Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins.

Affiliation:
Program in Neuroscience, Uniformed Services University of the Health Sciences , Bethesda, MD , USA.

ABSTRACTProtein citrullination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS), and prion disease. Citrullination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citrullination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up--regulated protein citrullination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders, including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically induced calcium excitotoxicity was shown to activate the citrullination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type, and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.